Platelets are anucleate cells derived from megakaryocytes (megs) that serve as critical components of hemostasis and thrombus formation, and mediate aspects of inflammation, immunity, and angiogenesis. In the adult, all platelets are derived from hematopoietic stem cells (HSCs). We previously discovered in the mouse embryo that the meg lineage is specified several days before HSC emergence as embryonic (pre- HSC) megakaryopoiesis. We have also determined that embryonic megs have limited polyploidization and generate extremely large platelets with small a-granules. Thrombopoiesis in human neonates is also characterized by limited polyploidization and rapid cytoplasmic maturation. Endoreplication is regulated in part by the Cip/Kip family of cell cycle inhibitors. Our preliminary studies indicate that embryonic, but not adult, platelets express high levels of p57 (Kip2). In Aim 1, we will further define the differences between embryonic, fetal and adult megakaryopoiesis and test the hypothesis that differences in meg endoreplication are regulated, in part, by the differential expression of Cip/Kip family members. Our preliminary studies of primary embryonic platelets indicate that they are effectively activated by thrombin but markedly less so by ADP. These functional studies correlate with the differential upregulation of PAR1 and down-regulation of P2Y12 in primary embryonic versus adult platelets. In Aim 2 studies, we will test the hypothesis that embryonic platelets have intrinsic functional differences in activation and clot formation when compared to their adult counterparts. An understanding of hematopoietic ontogeny is particularly relevant to the generation of blood cells from embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, which carry the potential to serve as an important source of cell-based therapies. We hypothesize that ES cell-derived megs and platelets will have predominantly embryonic characteristics. This hypothesis will be tested by comparing ES cell-derived meg maturation and platelet function with that of primary embryonic cells. This proposed research builds upon our studies of meg ontogeny and platelet emergence in the murine embryo and establishes a foundation for the development of clinically useful cell-based therapies from ES/iPS cell sources.